All patents, patent applications and references cited in this specification are incorporated herein by reference.
Carboxyl bearing polymers have found a wide usage in food, drugs, and industrial applications. A few well known examples include the carboxymethyl (‘CM’) polysaccharides CM-cellulose, CM-dextran, and CM-arabinogalactan (U.S. Pat. No. 5,981,507). The CM polysaccharides are produced from a reaction of the polysaccharide with haloacetic acids in base.
Carboxyl bearing polymers have been used in the synthesis of magnetic nanoparticles. Carboxylated dextrans of two major types have been used in the synthesis of superparamagnetic iron oxide nanoparticles. Carboxydextrans have a single terminal carboxyl group on each dextran molecule obtained by treatment with base (Hasegawa U.S. Pat. No. 4,101,435; Hasegawa U.S. Pat. No. 5,424,419, column 2, line 17). Carboxymethylated dextrans have numerous carboxymethyl groups attached per mole of dextran by reaction of alkyl halogenated acids in base (Maruno U.S. Pat. No. 5,204,457; Groman WO 00/61191).
Magnetic nanoparticles used for the attachment of biomolecules have been described by Molday (U.S. Pat. No. 4,452,773). A dextran coated magnetic nanoparticle is formed and then treated with periodate to produce aldehyde groups. The aldehydes react with amino groups on a biological molecule, to form a Schiff base. The Schiff base maybe stabilized by treatment of with a reducing agent like sodium borohydride. After treatment with a reducing agent a methylene amino linker connects the biomolecule to the nanoparticle. As shown in FIG. 2B, there are no peptidyl bonds in such linkages. A drawback of this method is the difficulty controlling the number and position of amino groups on the biomolecule that are available to react with the reactive aldehyde groups on the nanoparticle.
Other methods of attaching biomolecules to nanoparticles also use the reactivity of the aldehyde group. Rembaum and coworkers have utilized this approach, synthesizing nanoparticles with glutaraldehyde (U.S. Pat. No. 4,438,239; U.S. Pat. No. 4,369,226).
The development of amine functionalized crosslinked iron oxide nanoparticle (“amino-CLIO”, FIG. 2) by one of the inventors has proven to be an excellent method of synthesizing magnetic particle-biomolecule conjugates. Amino-CLIO is prepared by first synthesizing a dextran coated magnetic nanoparticle, followed by crosslinking the dextran with epichlorohydrin. Finally the amine groups are incorporated by reacting the dextran with ammonia (see Josephson et al, (1999) Bioconjug Chem 10, 186-91; Josephson et. al (2001) Angwandte Chemie 40, 3204-3206).
Amino-CLIO is an excellent label for the attachment of biomolecules, and for the synthesis of magnetic nanoparticle-biomolecule conjugates, for two reasons. First it provides an amine group for reaction with many bifunctional conjugation reagents that consist of N-hydroxysuccinimide esters that react first with an amine group and have a second group that reacts with sulfhydryl groups on a biomolecule. Examples of these bifunctional conjugating reagents are SPDP, SIA, SMCC and MBS. These reagents are available commercially (Pierce Chemical or Molecular Biosciences). Examples of biomolecules that have been attached to amino-CLIO include peptides (Josephson et al, (1999) Bioconjug Chem 10, 186-91), oligonucleotides (Josephson et. al (2001) Angwandte Chemie 40, 3204-3206) and proteins (Hogemann et al. (2000). Bioconjug Chem 11, 941-6). Second, amino-CLIO is highly stable due to the fact that the crosslinking forms a shell of dextran around a core of iron oxide. This allows storage of either amino-CLIO or bioconjugates based on amino-CLIO under a wide range of conditions (temperature, pH, ionic strength). By covalently joining polymeric molecules of the coating, crosslinking is associated with a pronounced increase in the molecular weight of the polymeric coating.
This amino-CLIO based chemistry has one major drawback, however, which arises precisely because of the extraordinary stability achieved by using a crosslinked-stabilized dextran on the nanoparticle surface. For human parenteral applications, such as for a magnetic label for targeted MR contrast agents, the degradation or elimination of the agent, including the coating, is required. However, when the iron oxide of an amino-CLIO based MR contrast is dissolved or biodegraded, the crosslinked dextran remains as a non-degradable sphere of polysaccharide. Similarly, non-degradability occurs with micron-sized magnetic microspheres where iron oxide is entrapped in a non-biodegradable polymeric shell (see U.S. Pat. No. 4,654,267; U.S. Pat. No. 5,512,439).
CM-polymers can also function as starting materials for the synthesis of drugs conjugates or for the attachment of various biological molecules. As drug conjugates, CM-arabinogalactan, CM-dextran and polyvinyl alcohol were used as carriers for nucleotide analogues (U.S. Pat. No. 5,981,507). The carboxyl groups were converted to primary amino groups by reaction with diamines. Biological molecules like araAMP were then attached to the primary amine of the aminated arabinogalactan, see Josephson, et al. (1996) Antivir Ther 1, 147-56 and U.S. Pat. No. 5,478,576.
In these examples, the CM-polymers, such as CM-arabinogalactan, exist as macromolecules in solution, which allows conditions to be employed that insure the nearly quantitative conversion of carboxyl groups to amino groups. The absence of protected carboxyl groups allows essentially all carboxyl groups to be chemically reactive.
There is a need for a improved magnetic nanoparticles to which biomolecules can be attached for use in cell sorting applications, in vitro assays, and which can be used as an intravenously administerable, MR contrast agents. The ideal nanoparticle must have a surface chemistry amenable to the efficient attachment of biomolecules with retention of their biological activity. It must be highly stable in vitro, both before and after the attachment of biomolecules. Yet it must be labile or degradable in vivo, with the utilization or elimination of all of its components.